Universal RHD genotyping in fetuses

Abstract

Is effective, and could dramatically reduce unnecessary anti-RhD prophylaxis

Non-invasive detection of fetal RHD status using maternal plasma is one of the few real advances in fetal medicine or obstetrics in recent years. DNA amplification of one or more region of the RHD gene can predict the fetal genotype with an accuracy of almost 99%.^{1 2} This means that invasive procedures (amniocentesis and chorionic villus sampling)—which carry a small but important risk of pregnancy loss and may also increase the risk of sensitisation in pregnancies at risk from fetal haemolytic disease—can be abandoned.

Non-invasive ascertainment of the fetal RHD genotype (and other red cell antigens) is now used routinely in the United Kingdom and elsewhere in the world.³ The technology, although labour intensive, is relatively simple and very reliable. In the accompanying study, Finning and colleagues assess the feasibility of applying a high throughput method for predicting RhD phenotype from fetal DNA in the plasma of pregnant women who are RhD negative.⁴

The incidence of RHD alloimmunisation has fallen greatly since the introduction of anti-RhD prophylaxis. In the 1960s prophylaxis was given in the postpartum period, and more recently after sensitising events and during the antenatal period. Antenatal prophylaxis is now offered to all RHD negative women in the UK and most developed countries regardless of the fetal genotype. But this approach has problems. Anti-D immunoglobulin is produced from pooled plasma taken from RHD negative male donors who have been injected with RhD positive red cells. Although the plasma is purified, the risk of infection remains. Outbreaks of hepatitis C have occurred, but with modern techniques the risk is minimal, although the possibility of infection with a viral or prion agent remains. Anti-RhD immunoglobulin is also in short supply and expensive, and the logistics of giving one or two doses antenatally is labour intensive and expensive. These injections are also painful and inconvenient, and many mothers have difficulty in accepting their need, especially as they are given without regard for fetal RHD status.

Currently, non-invasive fetal RHD genotyping is usually available only to women who have been sensitised—indicated by the detection of anti-D antibodies in an antenatal sample. After confirmation of the fetal genotype, only women carrying an RHD positive fetus are monitored for signs of fetal anaemia. Women with an RHD negative fetus can be discharged to routine obstetric care.

In their prospective study, Finning and colleagues assess high throughput RHD genotyping of fetal DNA in maternal plasma at 28 weeks—just before the first dose of antenatal anti-D injection—in about 1800 RHD negative women. The correct fetal genotype was predicted in almost 96% of cases, with a small false negative rate of 0.16%. The results were either inconclusive or unobtainable in only 3.4% of cases. These findings are important for two reasons. Firstly, they demonstrate the reliability of the automated technique and the feasibility of large scale antenatal testing. Secondly, if this approach was used for all RHD negative women, only 2% of these women would receive unnecessary anti-D prophylaxis, compared with 38% using the current system. This is a substantial reduction of an antenatal intervention.

The benefits of wider implementation of antenatal fetal genotyping are obvious. Antenatal prophylaxis can be given to those women who actually need it. The need for anti-RhD prophylaxis after potential sensitising events can also be eliminated if the fetus is definitely not at risk. This approach may also have financial implications. Although Finning and colleagues’ study did not assess costs, mass testing would probably reduce costs. The costs of testing everyone need to be balanced against the costs of giving immunoglobulin to everyone, but again mass testing is likely to result in a net saving by reducing pharmacy and manpower costs.

The consequences of a false negative result are important because withholding prophylaxis could potentially result in alloimmunisation and put the fetus and subsequent pregnancies at risk of haemolytic disease. In this study, only three samples (0.16%) gave false negative results. However, these samples were analysed more than 14 days from collection because of problems with transport. This delay would reduce the accuracy of the results because the amount of maternal DNA relative to fetal DNA increases in older samples, probably as a result of lysis of maternal cells in the specimen. The authors point out that under normal laboratory testing criteria these samples would have been discarded.

The consequences of a false positive result are far less detrimental. Some women will receive unnecessary anti-RhD prophylaxis as they do now, but the numbers would be small. Strict adherence to protocol would minimise the risks of false negative, false positive, or inconclusive results.

Although this study looked at samples taken from women at 28 weeks’ gestation, many potentially sensitising events occur before this time. If fetal genotyping is performed only in the third trimester many women will still receive unnecessary anti-RhD prophylaxis. Furthermore, prophylaxis after invasive first or early second trimester procedures will still be required. The goal of non-invasive testing is to determine fetal genotype as early in pregnancy as possible. Earlier gestation feasibility studies are in progress.

Universal fetal genotyping of all RHD negative women is the logical extension of a service that is already readily available. Automated techniques should make mass testing easier and cost effective, and they should minimise the risks of receiving an unnecessary blood product. If these techniques are shown to be as reliable earlier in pregnancy the arguments for recommending universal testing will be compelling.